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Committed to the advancement of Clinical & Industrial Disinfection & Microbiology
VOLUME - I
ISSUE - III
MAY-JUN 2008
Editorial
Contents
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Editorial
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Mini review
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Encyclopedia
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Current Trends
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In Profile
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Relax Mood
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Bug of the Month 11
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Did you Know
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Best Practices
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In Focus
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For Private Circulation Only
Nosocomial infections are defined as infections that are not present or
incubating in a patient at the time of admission to a hospital or health care facility. The
Centers for Disease Control and Prevention (CDC) estimates that nosocomial
infections occur in 5% to 10% of the patients annually. These types of infections also
contribute to deaths and increase the cost of the health care system per year.
Nosocomial infections continue to be a major concern for the health care industry.
National Nosocomial Infections Surveillance (NNIS) is a voluntary, hospital-based
reporting system established to monitor hospital-acquired infections and to guide the
prevention efforts of infection control practioners. In Mini Review we have included a
study of Nosocomial Infection and the control program.
Here we have again come up with an interesting topic in our Current Trends
section. Earlier povidone iodine was widely used as antiseptics but it has some
drawbacks. Now we have explained why Chlorhexidine gluconate is a preferred
antiseptic over povidone iodine. Pasteurization is a process, which is frequently used
in beverage and dairy industry and we have enlightened the biography of the
discoverer of the process, Louis Pasteur in our In Profile section. Bug of the Month is
focused on Salmonella species, which is causative agent of typhoid fever. It also causes
food borne infections. Antigenic structure of Salmonella is an important criterion,
discussed in this issue. As you are aware that antibiotic sensitivity testing is one of the
most important tests performed by microbiologist in clinical segment. McFarland
Standard No. 0.5 (turbidity standard) is mainly used in Antibiotic sensitivity testing for
standardization of bacterial suspension and you can know lot more about it in our Did
You Know section.
Autoclave is most widely used for sterilization of culture media and surgical
supplies. It is the most reliable method among all the moist heat sterilization methods.
Autoclave is a pressurized device designed to heat aqueous solutions above their
boiling point to achieve sterilization. We have described all the details of operation,
validation, and safety instructions about autoclave in our Best Practices segment.
Some more interesting matters are there for you in Encyclopedia, Relax Mood and In
Focus pages.
We would like to thank all our valued readers for appreciating and
encouraging the previous two efforts. We want more support from you to make
Journal of Hygiene Science more successful. You can communicate your views to us
through fax or e-mail.
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MAY-JUN 2008
A Study of Nosocomial Infection
and Control Program
Nosocomial Infection
The term nosocomial infection or hospital-acquired infection is
applied to any clinical infection that was neither present nor was
in its incubation period when the patient entered the hospital.
Nosocomial infections may also make their appearance after
discharge from the hospital, if the patient was in the incubation
period at the time of discharge. It is also known as Healthcare
associated infections. Within hours after admission, a patient's
flora begins to acquire characteristics of the surrounding bacterial
pool. Most infections those become clinically evident after 48
hours of hospitalization are considered hospital-acquired.
Infections that occur after the patient's discharge from the
hospital can be considered to have a nosocomial origin if the
organisms were acquired during the hospital stay.
· Compromised nutritional or immune status
· Use of indwelling catheters
· Failure of health care workers to wash their hands between
patients or before procedures
· Prevalence of antibiotic-resistant bacteria from the overuse of
antibiotics
The sources of the infecting organism are mainly from another
patient or a member of hospital staff, or from the inanimate
environment in the hospital and sometimes from patient's own
flora. Any type of invasive (enters the body) procedure can
expose a patient to the possibility of infection. Some common
procedures that increase the risk of hospital-acquired infections
include:
· Urinary bladder catheterization
· Respiratory procedures such as intubations or mechanical
ventilation
· Surgery and the dressing or drainage of surgical wounds
· Gastric drainage tubes into the stomach through the nose or
mouth
· Intravenous (IV) procedures for delivery of medication,
transfusion, or nutrition
Historical Perspective
The modern era of infection control began in the early 1950s; the
recognition and the awareness that the provision of medical and
nursing care in an institutional setting (e.g., a hospital) could
result in an increased risk for the acquisition of infection occurred
more than 100 years ago. In the 1840s, Dr. Ignaz Philip
Semmelweiss was concerned about the incidence of puerperal
fever in obstetrics ward and its related mortality. He postulated
that the puerperal fever that affected so many parturient women
was caused by the transmission through the hands of medical
personnel. Then he represented the first evidence indicating that
cleansing heavily contaminated hands with an antiseptic between
patient contacts may reduce the healthcare associated
transmission.
Mode Of Transmission
Microorganisms are transmitted in hospitals by several routes,
and the same microorganism may be transmitted by more than
one route. There are five main routes of transmission -- contact,
droplet, airborne, common vehicle, and vectorborne.
(a) Contact Transmission- It is the most important and frequent
mode of transmission of nosocomial infections and divided into
two subgroups: direct contact transmission and indirect contact
transmission.
· Direct Contact Transmission - This involves direct physical
transfer between a susceptible host and an infected or
colonized person, such as occurs between patient and hospital
personnel when personnel are turning patients, giving baths,
changing dressings, or performing other procedures requiring
direct personal contact. Taking care of patients generally
involves some direct contact. Direct contact transmission also
can occur between two patients, with one serving as the
source of the infectious microorganisms and the other as a
susceptible host.
· Indirect Contact Transmission - This involves personal
contact of the susceptible host with a contaminated
intermediate object, usually inanimate, such as instruments,
dressings, or other infective material. If proper care is not
taken, personnel can contaminate objects when assembling or
Causes Of Nosocomial Infections
All hospitalized patients are at risk of acquiring an infection from
their treatment or surgery. Some patients are at greater risk than
others, especially young children, the elderly, and persons with
compromised immune systems. The National Nosocomial
Infection Surveillance System database compiled by the CDC
shows that the overall infection rate among children in intensive
care is 6.1%, with the primary causes being venous catheters and
ventilator-associated pneumonia. The risk factors for hospitalacquired infections in children include parenteral nutrition (tube
or intravenous feeding), the use of antibiotics for more than 10
days, use of invasive devices, poor postoperative status, and
immune system dysfunction. Other risk factors that increase the
opportunity for hospitalized adults and children to acquire
infections are:
· A prolonged hospital stay
· Severity of underlying illness
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handling critical equipment (such as respiratory therapy
equipment, pressure-monitoring devices, cardiac bypass
pumps) or during other procedures that involve inanimate
objects.
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(b) Droplet Transmission - Infectious agents may come in contact
with the conjunctivae, nose, or mouth of a susceptible person as a
result of coughing, sneezing, or talking by an infected person.
These droplets are propelled through the air for a short distance
(approx. 3 feet). Generally influenza virus is transmitted by
droplet transmission.
Urinary Tract Infection - Urinary tract infection (UTI) is the most
common type of hospital-acquired infection and has been shown
to occur after urinary catheterization. Catheterization is the
placement of a catheter through the urethra into the urinary
bladder to empty urine from the bladder; or to deliver medication,
relieve pressure, or measure urine in the bladder; or for other
medical reasons. Normally, a healthy urinary bladder is sterile,
with no harmful bacteria or other microorganisms present.
Although bacteria may be in or around the urethra, they normally
cannot enter the bladder. A catheter, however, can pick up bacteria
from the urethra and give them an easy route into the bladder,
causing infection. Bacteria from the intestinal tract are the most
common type to cause UTIs. Patients with poorly functioning
immune systems or who are taking antibiotics are also at
increased risk for UTI caused by a fungus called Candida. The
prolonged use of antibiotics, which may reduce the effectiveness
of the patient's own immune system, has been shown to create
favorable conditions for the growth of this fungal organism.
(c) Air Borne Transmission - It occurs by dissemination of either
droplet nuclei (residue of evaporated droplets that may remain
suspended in the air for long periods of time) or dust particles in
the air containing the infectious agent. Organisms carried in this
manner are then inhaled by or deposited on the susceptible host.
Microorganisms transmitted by airborne transmission include
Mycobacterium tuberculosis and varicella viruses.
(d) Common Vehicle Transmission - It applies microorganisms
transmitted to the host by contaminated items such as food, water,
medications, devices, and equipment. Food borne illnesses are
frequently caused by common vehicle transmission and occur
when a number of people ingest the same contaminated food (e.g.
staphylococcal food poisoning) or contaminated water.
Respiratory Tract Infection - Pneumonia is another type of
hospital-acquired infection. Bacteria and other microorganisms
are easily introduced into the throat by treatment procedures
performed to treat respiratory illnesses. Patients with chronic
obstructive lung disease are especially susceptible to infection
because of frequent and prolonged antibiotic therapy and longterm mechanical ventilation used in their treatment. The infecting
microorganisms can come from contaminated equipment or the
hands of health care workers as procedures are conducted such as
respiratory intubation, suctioning of material from the throat and
mouth, and mechanical ventilation. Once introduced through the
nose and mouth, microorganisms quickly colonize the throat area.
This means that they grow and form a colony, but have not yet
caused an infection. Once the throat is colonized, it is easy for a
patient to aspirate the microorganisms into the lungs, where
infection develops that leads to pneumonia.
(e) Vector Borne Transmission - It is of greater concern in
developing countries for example, mosquito-transmitted malaria.
Since agent and host factors are more difficult to control,
interruption of the chain of infection in the hospital is directed
primarily at transmission.
Types Of Hospital Acquired Infections
The most common types of nosocomial infections that could
occur in the hospital set up are:
· Surgical wound and other soft tissue infections
· Urinary tract infections
· Respiratory infections
· Gastroenteritis
· Meningitis
Epidemiology and Transmission of Infections among
Hospital Personnel and Patient
Bacteria, viruses, fungi, or parasites can cause hospital-acquired
infections. These microorganisms may already be present in the
patient's body or may come from the environment, contaminated
hospital equipment, health care workers, or other patients.
Depending on the causal agents involved, an infection may start
in any part of the body. Almost any transmissible infection may
occur in the community at large or within the hospital and can
affect both personnel and patients. Transmission can occur from
personnel to patient as well as from patient to personnel.
Some of the following diseases are transmitted mostly from
Surgical site infection - Invasive surgical procedures increase a
patient's risk of getting an infection by giving bacteria a route into
normally sterile areas of the body. An infection can be acquired
from contaminated surgical equipment or from the hands of
healthcare workers. Following surgery, the surgical wound can
become infected from contaminated dressings or the hands of
healthcare workers who change the dressing. Other wounds can
also become easily infected, such as those caused by trauma,
burns, or pressure sores that result from prolonged bed rest or
wheel chair use. Factors most consistently associated with an
increased incidence of postoperative infections are:
For Private Circulation Only
Over 60 years, of age
Preoperative stay in hospital
Long duration of the surgical procedure
Pre-existing infection at the site of the wound
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health care personnel to patient and vice versa.
· Acute Diarrhea - Various agents may cause diarrhea in
patients and hospital personnel. Salmonella, Shigella, and
Campylobacter species are among the common bacterial
enteric pathogens. Rotavirus and other viral agents can cause
gastroenteritis. Giardia lamblia and other protozoa are also
frequent causes of diarrhea. Any of these agents may be
nosocomially transmitted via the hands of personnel who are
infected.
· Hepatitis - Viral hepatitis has long been recognized as a
nosocomial hazard. The agents that most commonly cause
viral hepatitis are hepatitis A virus (HAV) and hepatitis B
virus (HBV). Primarily the fecal-oral route transmits
Hepatitis A. Fecal excretion of HAV is greatest during the
incubation period of disease before the onset of jaundice.
Once disease is clinically obvious, the risk of transmitting
infection is decreased. Some patients admitted to the hospital
with hepatitis A may still be shedding virus and are potentially
infective. Personnel can help protect themselves and others
from infection with HAV by always maintaining good
personal hygiene, practicing thorough hand washing at all
times.
Most nosocomial cases of hepatitis B unrelated to the
transfusion of blood or blood products occur in hospital
personnel rather than patients. Transmission occurs by
parenteral or mucosal exposure to HBsAg-positive blood
from persons who are carriers or have acute HBV infection.
Often carriers of HBsAg and persons with acute infections are
unrecognized and are therefore not known to be infective.
Since droplets from the patient's mouth reach the face of the
dentist during certain procedures, dentists might consider
protecting their eyes, nose, and mouth from such exposure by
using masks and protective eyewear. They can prevent direct
contact with infective material in the mouth by routinely
wearing gloves during dental procedures.
· Herpes simplex viruses (HSV) can be transmitted among
personnel and patients through either primary or recurrent
lesions or through secretions. Many sites can become infected,
exposed areas of skin are most likely to be involved,
particularly when minor cuts, abrasions, or other skin lesions
are present. Direct contact with lesions or infected secretions
is the principal mode of spread. So it is a distinct hazard for
nurses, anesthesiologists, dentists, respiratory care personnel,
and other personnel who may have direct (usually hand)
contact with either oral lesions or respiratory secretions from
patients. Personnel can protect themselves from such
infections by 1) avoiding direct contact with lesions, 2)
wearing gloves on both hands or using "no-touch" technique
for all contact with oral or vaginal secretions, and 3) thorough
hand washing after patient contact.
· Staphylococcus infection frequently occurs in human. In
nosocomial transmission, there are two sources: a person with
a lesion or an asymptomatic carrier. Persons with skin lesions
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due to S. aureus are most likely to disseminate these
organisms. Direct contact is the major route of transmission.
In the hospital, infection is most likely to occur when a patient
has unsuspected pulmonary or laryngeal TB, has bacilli-laden
sputum or respiratory secretions, and is coughing or sneezing
into air that remains in circulation. The best ways to protect
others from a patient with TB are to maintain a high index of
suspicion for TB and to institute appropriate precautions.
Nosocomial transmission of varicella- zoster infection among
personnel and patients is well recognized. Appropriate
isolation of hospitalized patients with known or suspected
varicella or zoster can reduce the risk of transmission to
personnel. Varicella is transmitted primarily via airborne
spread by small particle aerosols (droplet nuclei) and by large
particles (droplets).
Viral respiratory infections are common problems for
infection control programs. The three chief mechanisms of
transmission of respiratory viruses are 1) small-particle
aerosols (droplet nuclei), 2) large particles (droplets), and 3)
inoculation of viruses after direct contact with infective areas
or materials. Different respiratory viruses may vary in the way
in which they are transmitted. Small-particle aerosols are
produced by talking, sneezing, or coughing and may transmit
infection over a considerable distance. Large particles
(droplets) are produced by sneezing and coughing and require
close person-to-person contact for transmission. Person-toperson transmission can also occur by contaminating the
hands by direct contact with infective areas or materials, then
transferral of infective virus to mucous membranes of a
susceptible person. Self-inoculation can also occur in this way.
The nose and eyes, rather than the mouth, appear to be
important portals of entry.
Pediatric patients appear to be at particular risk for
complications from nosocomial respiratory tract infections.
Infection in the elderly, patients with chronic underlying
illness, and immunocompromised patients may also be
associated with significant morbidity. Thus, it may be prudent
to exclude personnel with viral respiratory infections from the
care of these high-risk patients. Because large numbers of
personnel may have viral respiratory illnesses during the
winter, it may not be possible to restrict all such personnel
from taking care of patients not in high-risk groups. Careful
hand washing before patient contact is essential in preventing
transmission. Masks might be beneficial in preventing
transmission by large droplets from personnel to patients
upon close contact. However, masks probably will not
completely protect personnel from patients with respiratory
illnesses because large particles and aerosols may still reach
the eyes, and self-inoculation from contaminated hands can
still occur by touching the eyes.
Personnel may be exposed to patients with cytomegalovirus
(CMV) infection, but the risk of acquiring CMV infection
from patients appears to be small. There are two principal
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reservoirs of CMV in the hospital: 1) infants infected with
CMV and 2) immunocompromised patients, such as
oncology patients and those undergoing kidney or bone
marrow transplant. Infection appears to be acquired only
through intimate, direct contact with an excreter of CMV or
contact with contaminated secretions. Virus can be shed in the
urine, saliva, respiratory secretions, tears, feces, breast milk,
semen and cervical secretions. A practical approach to reduce
the risk of infection with CMV is to stress careful hand
washing after all patient contacts and avoiding contact with
areas or materials that are potentially infective.
· Nosocomial transmission of Neisseria meningitidis to
hospital personnel taking care of patients with
meningococcemia, meningococcal meningitis, or lower
respiratory infections is uncommon. In rare instances
transmission to personnel from patients with
meningococcemia or meningococcal meningitis has occurred
through intensive direct contact with the infected person and
direct contact with respiratory secretions without use of
proper precautions. The most likely mode of spread from a
person with infections at these sites is by large droplet
secretions.
· Pertussis, caused by Bordetella pertussis, is highly
communicable. Nosocomial transmission of pertussis has
been reported infrequently. Although infection occurs less
commonly in adults and may be limited to mild respiratory
illness, personnel with pediatric patient contact may be
involved in transmission of pertussis to patients.
· Scabies is a disease caused by infestation with the mite
Sarcoptes scabiei. It is transmitted in hospitals primarily
through intimate direct contact with an infested person, even
when high levels of personal hygiene are maintained.
Transmission to personnel has occurred during activities such
as sponge-bathing patients or applying body lotions.
hospital personnel fail to follow basic infection control, such as
hand washing between patient contacts. In ICUs, asepsis is often
overlooked in the rush of crisis care. Third, patients in hospitals
are increasingly immunocompromised. The shift of surgical care
to outpatient centers leaves the sickest patients in hospitals,
which are becoming more like large ICUs. This shift has led to the
greater prevalence of vascular access associated bloodstream
infections and ventilator-associated pneumonias.
Control Measures
The CDC (1985) on the efficacy of nosocomial infection control
(SENIC) showed beyond doubt that increase in surveillance
activities is able to directly bring down the rates of nosocomial
infections. It is only too well known that nosocomial infections
are most prevalent in certain high risk areas such as the intensive
care renal dialysis and organ transplant units, burns ward, cancer
ward, operation theatres, post-operation theatres, postoperative
ward nursery and the geriatric ward. Hospitals take a variety of
steps to prevent nosocomial infections, including:
· Sterilization should be done of all reusable equipment such as
ventilators, humidifiers, and any devices that come in contact
with the respiratory tract.
· Handwashing with high level disinfectants frequently is
called the single most important measure to reduce the risks of
transmitting microorganisms from one person to another or
from one site to another on the same patient. Washing hands as
promptly and thoroughly as possible between patient contacts
and after contact with blood, body fluids, secretions,
excretions and equipment or articles contaminated by them is
an important component of infection control and isolation
precautions. Although handwashing may seem like a simple
measure, it is often not used or hand washing is performed
incorrectly. Healthcare settings must continually remind
practitioners and visitors to wash their hands thoroughly.
· Strict attention should be given to aseptic (sterile) technique
in the performance of procedures, including use of sterile
gowns, gloves, masks, and barriers. In addition to
handwashing, gloves play an important role in reducing the
risks of transmission of microorganisms. Gloves are worn for
three important reasons in hospitals. First, gloves are worn to
provide a protective barrier and to prevent gross
contamination of the hands when touching blood, body fluids,
secretions, excretions, mucous membranes, and nonintact
skin; the wearing of gloves in specified circumstances to
reduce the risk of exposures to blood borne pathogens.
Second, gloves are worn to reduce the likelihood that
microorganisms present on the hands of personnel will be
transmitted to patients during invasive or other patient-care
procedures that involve touching a patient's mucous
membranes and nonintact skin. Third, gloves are worn to
reduce the likelihood that hands of personnel contaminated
with microorganisms from a patient or a fomite can transmit
these microorganisms to another patient. Wearing gloves does
Reasons of Emerging Nosocomial Infections
Three major forces are involved in nosocomial infections. The
first is antimicrobial use in hospitals and long-term care facilities.
The increased concern about gram-negative bacilli infections in
the 1970s to 1980s led to increased use of cephalosporin
antibiotics. As gram-negative bacilli became resistant to earlier
generations of cephalosporin antibiotics, newer generations were
developed. Widespread use of cephalosporin antibiotics is often
cited as a cause of the emergence of enterococci as nosocomial
pathogens. About the same time, MRSA, perhaps also in response
to extensive use of cephalosporin antibiotics, became a major
nosocomial threat. Widespread empiric use of vancomycin, as a
response to concerns about MRSA and for treatment of vascular
catheter associated infection by resistant coagulase-negative
staphylococci, is the major initial selective pressure for VRE. Use
of antimicrobial drugs in long-term care facilities and transfer of
patients between these facilities and hospitals have created a large
reservoir of resistant strains in nursing homes. Second, many
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not replace the need for handwashing, because gloves may
have small, non-apparent defects or may be torn during use,
and hands can become contaminated during removal of
gloves.
Use of an antibacterial-coated venous catheter can destroy
bacteria before they get into the blood stream. Use of silver
alloy-coated urinary catheters can destroy bacteria before
they migrate up into the bladder.
Frequent changing of dressings for wounds and use of
antibacterial ointments under dressings can prevent the
spread of infection.
Isolation precautions are designed to prevent transmission of
microorganisms by common routes in hospitals. Because
agent and host factors are more difficult to control,
interruption of transfer of microorganisms is directed
primarily at transmission.
Sterilization of medical instruments and equipment should be
done to prevent contamination.
MAY-JUN 2008
· An infection control practitioner should be employed for
every 200 beds.
· The general use of antibiotics should be reduced to encourage
better immune response in patients and reduce the cultivation
of resistant bacteria.
References
Robert J. Sherertz and Felix A. Sarubbi. 1983. A Three-Year
Study of Nosocomial Infections Associated with Pseudomonas
aeruginosa. Journal of Clinical Microbiology. 18(1): 160-164.
T. Grace Emori and Robert P. Gaynes. 1993. An overview of
nosocomial infections, including the role of the microbiology
laboratory. Clinical Microbiology Reviews. 6(4): 428-442.
Vale´rie Livrelli et al. 1996. Adhesive Properties and Antibiotic
Resistance of Klebsiella, Enterobacter, and Serratia Clinical
Isolates Involved in Nosocomial Infections. Journal of Clinical
Microbiology. 34(8): 1963-1969.
Encyclopedia
(including particularly Mycobacterium tuberculosis and Coxiella
burnetii). A newer method called flash pasteurization involves
shorter exposure to higher temperatures, and is claimed to be
better for preserving color and taste in some products.
Pasteurization is the process of heating liquids (milk and
milk products, beverages) for the purpose of destroying viruses
and harmful organisms such as bacteria, protozoa, molds and
yeasts. The process was named after its inventor, French scientist
Louis Pasteur. Unlike sterilization, pasteurization is not intended
to kill all pathogenic microorganisms in the food. Instead,
pasteurization aims to achieve a logarithimic reduction in the
number of viable organisms, reducing their number so they are
unlikely to cause disease. Commercial-scale sterilization of food
is not common, because it adversely affects the taste and quality
of the product. Pasteurization typically uses temperatures below
boiling since at temperatures above the boiling point for milk,
casein micelles aggregate irreversibly.
There are two main types of pasteurization used presently: high
temperature/short time(HTST) and ultra-high temperature
(UHT). In the HTST process, milk is heated to 71.7 ºC (161ºF) for
15-20 seconds. UHT processing hold the milk at a temperature
138ºC (280ºF) for 2 seconds. In vat pasteurization the milk is
heated 63ºC (145ºF) for 30 minutes. Pasteurization methods are
usually standardized and controlled by national food safety
agencies such as USDA. There are different standards for
different dairy products, depending on the fat content and the
intended usage. For example, the pasteurisation standards for
cream differ from the standards for fluid milk, and the standards
for pasteurizing cheese are designed to preserve the phosphatase
enzyme, which aids in cutting. The HTST pasteurization standard
was designed to achieve a 5-log reduction (0.00001 times the
original) in the number of viable microorganisms in milk. This is
considered adequate for destroying almost all yeasts, mold, and
common spoilage bacteria and also to ensure adequate
destruction of common pathogenic heat-resistant organisms
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Methods of Pasteurization
There are two basic methods of pasteurization, batch or
continuous.
Batch method
The batch method uses a vat pasteurizer, which consists of a
jacketed vat, surrounded by either circulating water, steam or
heating coils of water or steam. In the vat the milk is heated and
held throughout the holding period while being agitated. The
milk may be cooled in the vat or removed hot after the holding
time is completed for every particle.
Continuous Method
Continuous process method has several advantages over the vat
method, the most important being time and energy saving. For
most continuous processing, a high temperature short time
(HTST) pasteurizer is used. The heat treatment is accomplished
using a plate heat exchanger.
Significance
There are two distinct purposes for the process of milk
pasteurization:
§ Public Health Aspect- To make milk and milk products safe
for human consumption by destroying all bacteria that may be
harmful to health (pathogens).
§ Keeping Quality Aspect- To improve the keeping quality of
milk and milk products. Pasteurization can destroy some
undesirable enzymes and many spoilage bacteria. Shelf life
can be 7, 10, 14 or up to 16 days.
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Current Trends
Chlorhexidine Gluconate
A Superior Antiseptic than Povidone Iodine
skin. It is widely used in preoperative and postoperative skin
treatment. Despite the wide use of povidone iodine it also possess
properties unsuitable for practical application. Povidone iodine
has the following drawbacks:
§ It is deactivated in the presence of organic materials (such as
pus, blood and exudates) and it is cytotoxic to the fibroblasts
in stronger concentrations.
§ Intermediate antimicrobial action is slow.
§ Absorption of iodine can occur in young children, in people
with thyroid problems, and people with poor renal function.
§ Povidone iodine is not recommended for long term wound
cleansing.
§ It has no residual effect.
§ It can cause pain in open wounds and also cause allergic
reactions to some hypersensitive patients.
§ Its effects wear off after a relatively short time and it does
discolour the skin.
§ Bacterial resistance may occur mainly with Staphylococcus
aureus.
§ Povidone iodine disinfection is tedious and has to follow
multi step procedure.
An antiseptic is a substance that prevents or arrests the growth or
action of microorganisms either by inhibiting their activity or by
destroying them. The term is used especially for preparations
applied topically to living tissue. A skin antiseptic is a safe,
nonirritating, antimicrobial-containing preparation that prevents
overt skin infection.
John Pringle apparently first used the term "antiseptic" (Greek for
"against putrefaction") in 1750 to describe substances that
prevent putrefaction. As so often in the history of medicine a
change of practice depended on the persistence of one man. For
antiseptics, this man was John Lister. He chose phenol and
applied it vigorously in surgery. The effect of Lister's practices
was revolutionary and the antiseptic technique opened the way to
great surgical advances.
Salient Features Of Antiseptics
An antiseptic has following features:
1) Antiseptics bind readily to bacteria; the amount absorbed
increasing with an increasing concentration solution. The
most important site of absorption is the cytoplasmic
membrane.
2) The extent of killing of the bacteria is governed by three
principal factors: (a) concentration of the antiseptic, (b)
bacterial cell density, and (c) time of contact. The absorption
of a given amount of the compound per cell leads to the killing
of a definite fraction of the bacterial population in a chosen
time interval.
3) The necessary characteristic of antiseptics is their bactericidal
action, but there is often a low and rather narrow
concentration range in which their effect is bacteriostatic. At
these low concentrations, certain biochemical functions
associated with the bacterial membrane may be inhibited.
4) In the presence of higher concentrations of antiseptic and after
prolonged treatment, the compound usually penetrates the
cell and brings about extensive ill-defined disruption of
normal cellular functions.
Chlorhexidine - The 'preferred' skin antiseptic
Chlorhexidine was first synthesized in 1950 in the laboratories of
ICI Ltd. (England). It was found to possess a high level of
antibacterial activity, low mammalian toxicity and a strong
affinity for binding to skin and mucous membrane. These
properties led to the development of chlorhexidine principally as
a topical antiseptic for application to such areas as skin, wounds
and mucous membranes and for dental use.
Chlorhexidine is 1,6-di (4-chlorphenyl-diguanido) hexane, a
cationic bisbiguanide compound. Bisbiguanides are the primary
second generation antiplaque agents. It is a strong base, at
physiologic pH, is a large dicationionic molecule.
Chlorhexidine has a broad spectrum of antimicrobial activity
including a wide range of Gram positive and Gram negative
bacteria, yeasts, dermatophytes and some lipophylic viruses
including HBV and HIV. At relatively low concentrations, the
action of chlorhexidine is bacteriostatic and at higher
concentrations, it is rapidly bactericidal, with the actual levels
varied from species to species. The lethal process consists of a
series of related cytologic and physiologic changes, some of
which are reversible, that culminate in the death of the cell. The
sequence is thought to be as follows:
§ The rapid attraction toward the bacterial cell.
§ Specific and strong adsorption to certain phosphate
containing compounds on the bacterial surface.
Povidone Iodine - An earlier used Antiseptic
The first use of iodine in medical practice was as a remedy for
bronchocele. The first specific reference to the use of iodine in
wounds was made in 1839. Iodine was officially recognized by
the Pharmacopeia of the United States in 1830. By the late
nineteenth century iodine tinctures came into wide use as
microbicides. Later povidone iodine is used as a topical antiseptic
agent. Povidone iodine is water-soluble complex of iodine with
polyvinylpyrrolidone. It is applied as an antiseptic in the form of
solutions or ointments. It is used in cleansing and disinfecting the
For Private Circulation Only
7
Current Trends
§ Overcoming the bacterial cell wall exclusion mechanisms.
§ Attraction toward the cytoplasmic membrane.
§ Leakage of low molecular weight cytoplasmic components,
such as potassium ions and inhibition of certain membrane
bound enzymes, such as adenosyl triphosphatase.
§ Precipitation of the cytoplasm by the formation of complexes
with phosphated entities, such as adenosine triophosphated
entities, such as adenosine triphosphate and nucleic acids.
A bacterial cell is negatively charged, the nature of the ionogenic
groups varying with bacterial species. It has been shown that,
given sufficient chlorhexidine concentration and reaches a stable
equilibrium within 5 minutes. The rapid electrostatic attraction of
the cationic chlorhexidine molecules and the negatively charged
bacterial cell undoubtedly contributes to the rapid rate of killing
associated with chlorhexidine, although surface charge reversal
is secondary to cell death. Chlorhexidine molecules are thought
to compete for the negative sites on the peptidoglycan, thereby
displacing metallic cations. The bacterial cytoplasmic membrane
appears to be the important site of action. Several changes
indicative of damage to the cytoplasmic membrane have been
observed in bacterial populations treated with bacteriostaitic and
bactericidal levels of chlorhexidine. Leakage of cytoplasmic
contents is a classic indication of damage to the cytoplasmic
membrane, starting with low molecular molecules typified by
potassium ions. Sub lethally treated cells show a shrinkage or
plasmolysis of the protoplast.
Chlorhexidine also has depathogenizing effect. Chlorhexidine
inhibits the growth of the vegetative cells of spore forming
bacteria at relatively low concentrations and also inhibits the
spore germination. Chlorhexidine has good activity against
viruses with a lipid component in their coats or with an outer
envelope. These include many respiratory viruses, herpes, and
cytomegalovirus.
free of bacteria, thus preventing the escape of organisms into the
operation wound through the puncture in surgical gloves, which
occur frequently during operation. The procedure must eliminate
the transient organisms that are likely to be present on the skin and
reduce the resident flora to as low level as possible. The agent
should remain persistent on the skin to maintain the numbers of
survivors at this low level through out the course of the operation.
The use of 2-4% chlorhexidine as handwash is found to be
effective antiseptic in reducing the numbers of bacteria on the
hands of the surgical personnel and maintaining these low
numbers for several hours under gloves. A povidone iodine
handwash is less effective initially and allowed the numbers of
survivors on the hands to increase dramatically during the
course of an operation. Chlorhexidine handwash significantly
reduce a greater number of resident floras than povidone iodine
handwash.
Preoperative Whole Body Disinfection
A significant proportion of postoperative wound infections are
caused by microorganisms from the patient's own skin, which
may be derived from sites remote from that of the operation.
Chlorhexidine skin cleanser is found to reduce the skin floras
and maintain this reduction for up to 1 week. Preoperative
showering of patient with chlorhexidine is effective to control
the infection rate.
Hygienic Hand Disinfection
The major source of infectious organisms within the hospital is
the infected or heavily colonized patient, and the principal route
of transmission is via the hands of hospital personnel. Washing
hands with high level is therefore considered to be the single
most important measure to prevent nosocomial infection.
Other than these Chlorhexidine gluconate can also reduce the risk
for catheter-related blood stream infection by approximately
50% in hospitalized patients who require short-term
catheterization. Povidone iodine is not persistent and allows a
significant increase in bacterial numbers with time. So CDC has
recommended chlorhexidine gluconate solution to prevent
infections related to invasive devices. Chlorhexidine gluconate
solution can also be used in pharmaceutical industry as well as in
general purpose. Alcoholic chlorhexidine gluconate solution is
much more effctive antiseptic than povidone iodine. So it is time
for health practioners to shift from povidone iodine to more
effective chlorhexidine gluconate to control the infection rate.
Applications and Advantages of Chlorhexidine over
Povidone Iodine
Nowadays chlorhexidine is used in general medicine for skin
disinfection, surgical hand disinfection, preoperative whole body
disinfection, hygienic hand disinfection, urology, obstetrics and
gynecology, nasal cavity and throat, wounds and burns.
Skin disinfection
Chlorhexidine formulated in a detergent base is used extensively
for disinfection of the hands of surgeons and nurses and also for
whole body skin disinfection of patients undergoing surgery.
Surgeons and nurses for hand disinfection also use alcohol-based
chlorhexidine solutions with emollients. Alcohol based
chlorhexidine solutions are particularly suitable for final stage
skin preparation of the operation site; the area should be kept wet
for at least 2 minutes to achieve the maximal effect.
References
J. Barenfanger, C. Drake, J. Lawhorn and S.J. Verhulst. 2004.
Comparison of Chlorhexidine and Tincture of Iodine for Skin
Antisepsis in Preparation for Blood Sample Collection. J Clin
Microbiol. 42(5): 2216-2217.
Oliver Mimoz et. al. 2003. Chlorhexidine compared with
povidone-iodine as skin preparation before blood culture.
Journal of the American Medical Association. 289: 1274-1277.
Surgical Hand Disinfection
The objective of surgical hand disinfection is to render the skin
For Private Circulation Only
MAY-JUN 2008
8
In Profile
MAY-JUN 2008
conducted experiments that clearly indicated its correctness and
managed to convince most of Europe it was true.
The country's enormous production of silk had suddenly been
curtailed because of an epidemic illness among the silkworms.
Despite having no prior experience with animal biology, Pasteur
determined the silkworms were dying from two parasitic
infections. Pebrine, in which black spots and corpuscles are
generally, but not always, present on the worm. In such cases the
worms die within the cocoons. In the second type of disease,
flacherie, the worms exhibit no corpuscles or spots but fail to spin
cocoons. Suspecting that certain microscopic objects found in the
diseased silkworms (and in the moths and their eggs) were
disease-producing organisms, Pasteur experimented with the
separation of uninfected from infected silkworms, the cleanliness
of silkworm colonies, and controlled breeding. He concluded that
only in diseased and living eggs was the cause of the disease
maintained; therefore, selection of disease-free eggs was the
solution.
Pasteur also determined the natural history of anthrax, a fatal
disease of cattle. He proved that anthrax is caused by a particular
bacillus and suggested that animals could be given anthrax in a
mild form by vaccinating them with attenuated bacilli, thus
providing immunity from potentially fatal attacks.
Pasteur spent the rest of his life working on the causes of
various diseases - including septicemia, cholera, diphtheria, fowl
cholera, tuberculosis, smallpox and their prevention by means of
vaccination. He is best known for his investigations concerning
the prevention of rabies, otherwise known in humans as
hydrophobia. After experimenting with the saliva of animals
suffering from this disease, Pasteur concluded that the disease
rests in the nerve centers of the body; when an extract from the
spinal column of a rabid dog was injected into the bodies of
healthy animals, symptoms of rabies were produced. By studying
the tissues of infected animals, particularly rabbits, Pasteur was
able to develop an attenuated form of the virus that could be used
for inoculation. In 1885, a young boy and his mother arrived at
Pasteur's laboratory; the boy had been bitten badly by a rabid dog,
and Pasteur was urged to treat him with his new method. At the
end of the treatment, which lasted ten days, the boy was being
inoculated with the most potent rabies virus known; he recovered
and remained healthy. Since that time, thousands of people have
been saved from rabies by this treatment.
The French government honoured Pasteur with its highest
award, the Legion of Honour. Louis Pasteur died at Saint Cloud
on 28 September 1895 and was given a state funeral at the
Cathedral of Notre Dame and his body placed in a permanent
crypt at the Pasteur Institute. His work served as the springboard
for branches of science and medicine such as stereochemistry,
microbiology, bacteriology, virology, immunology, and
molecular biology. Moreover, his work has protected millions of
people from disease through vaccination and pasteurization.
Louis Pasteur
Birth: December 27, 1822
Death: September 28, 1895
Nationality: French
Known For: Germ theory of disease,
Pateurization Technique
Louis Pasteur, world-renowned French chemist and biologist,
who founded the science of microbiology, proved the germ
theory of disease, invented the process of pasteurization and
developed vaccines for several diseases, including rabies.
Pasteur was born in Dôle on December 27, 1822, the son of a
tanner, and grew up in the small town of Arbois. In 1847 he earned
a doctorate at the École Normale in Paris, with a focus on both
physics and chemistry. Becoming an assistant to one of his
teachers, he began research that led to a significant discovery. He
found that a beam of polarized light was rotated to either the right
or the left as it passed through a pure solution of naturally
produced organic nutrients, whereas when polarized light was
passed through a solution of artificially synthesized organic
nutrients, no rotation took place. If, however, bacteria or other
microorganisms were placed in the latter solution, after a while it
would also rotate light to the right or left.
After spending several years of research and teaching at Dijon
and Strasbourg, Pasteur moved in 1854 to the University of Lille,
where he was named professor of chemistry and dean of the
faculty of sciences. This faculty had been set up partly to serve as
a means of applying science to the practical problems of the
industries of the region, especially the manufacture of alcoholic
beverages. Pasteur immediately devoted himself to research on
the process of fermentation. He studied lactic and alcoholic
fermentation and showed that all fermentation is due to the
presence of microorganisms.
The souring of wine and beer had been a major economic
problem in France; Pasteur contributed to solving the problem by
showing that heating the starting sugar solutions to a high
temperature could eliminate bacteria. Pasteur achieved success
by slightly modifying the process used with the broth. Boiling the
wine would alter its flavour. Therefore, Pasteur heated the wine
enough to kill most of the microbes present without altering the
flavour. This process is later known as pasteurization.
Louis Pasteur demonstrated that the fermentation process is
caused by the growth of microorganisms, and that the growth of
microorganisms in nutrient broths is not due to spontaneous
generation. He exposed boiled broths to air in vessels that
contained a filter to prevent all particles from passing through to
the growth medium, and even in vessels with no filter at all, with
air being admitted via a long tortuous tube that would not allow
dust particles to pass. Nothing grew in the broths; therefore, the
living organisms that grew in such broths came from outside, as
spores on dust, rather than spontaneously generated within the
broth. This was one of the last and most important experments
disproving the theory of spontaneous generation. While Pasteur
was not the first to propose germ therapy, he developed it and
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References
J.R. Porter. 1961. Louis Pasteur: achievements and
disappointments. MMBR. 25: 389-403
9
Relax Mood
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Enjoy t
Thoughts to live by
§ Mary was discussing the various aspects and possible outcome
of the insurance policy with the clerk at the Insurance Agency.
During the discussion, she asked. "Suppose I take the life
insurance for my husband today for a million dollars, and
tomorrow he dies? What will I get?”
The clerk eyed her suspiciously and replied, "Probably a life
sentence."
§ The grand essentials of happiness are: something to do,
something to love and something to hope for. (Allan K.
Chalmers)
§ Hold fast to dreams, for if dreams die, life is a broken
winged bird that cannot fly. (Lanston Hughes)
§ The secret of success in life is for a man to be ready for
his opportunity when it comes. (Earl of Beaconsfield)
§ A physician, a civil engineer, and a consultant were arguing
about what was the oldest profession in the world.
The physician remarked, "Well, in the Bible, it says that God
created Eve from a rib taken out of Adam. This clearly required
surgery, and so I can rightly claim that mine is the oldest profession
in the world."
The civil engineer interrupted, and said, "But even earlier in the
book of Genesis, it states that God created the order of the heavens
and the earth from out of the chaos. This was the first and certainly
the most spectacular application of civil engineering. Therefore, fair
doctor, you are wrong: mine is the oldest profession in the world.”
The consultant leaned back in her chair, smiled, and then said
confidently, "Ah, but whom do you think created the chaos?”
§ Lucky is the man who is the first love of a woman, but
luckier is the woman who is the last love of a man.
§ Time is a companion that goes with us on a journey. It
reminds us to cherish each moment, because it will
never come again. What we leave behind is not as
important as how we have lived. (Captain Jean Luc
Picard).
Track your brain
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3. Glycocalyx- producing bacterial species that causes anthrax(initials). 5. The
genetic material of a bacterium. 6. A cluster of eight cocci. 7. Staining allows
one to determine the shape,________, and arrangement of bacterial cells. 8.
Contains the enzyme lysozyme for bacterial destruction. 10. The mosaic
model describes the structure of the cell membrane. 11. The cell membrane is
the structure for nutrient _____ into the cell cytoplasm. 13. It includes capsules
and slime layers. 15. Closed loop of DNA having the bacterial inheritance
characteristics. 18. Nucleic acid found in plasmids. 21. The important
chemical constituent of the bacterial cell wall. 23. Unit of measurement for
viruses(abbr). 24. Spiral bacteria with a flexible cell wall. 26. A short hair-like
projection for attachment and genetic transfer. 28. Bodies of RNA and protein
that function in protein systhesis. 30. Bacterium that occurs as a _______
sarcina (initials). 32. Closed loop of DNA apart from the chromosome in the
cytoplasm. 35. The slime of the capsule of A. viscolactis is________ positive
bacteria. 37. Teichoic acid is present in the cell walls of_______ positive
bacteria. 40. Flagella permit a bacterium to_______. 41. A monotrichous
bacterium has_____. 42. One of the disaccharides in the bacterial cell
wall(abbr). 43. The cell membrane has_______ important organic
constituents. 44. The name for the chromosomal region of a bacterium.
1. Bacterium (initials) that appears as a grape like cluster. 2. A pair of bacterial cocci is a_______ coccus. 3. A bacterial rod. 4. A curved rod that resembles a comma
under the microscope. 5. A polysaccharide existing as tangled fibers in the glycocalyx. 6. A thin, loosely bound glycocalyx is called the ______ layer. 9. About 40 percent
of the cell membrane consists of_______. 12. The side chains of peptidoglycan consist of_______ amino acids. 14. The presence of a capsule contributes to the ability
of a pathogen to cause_____ . 16. A capsule containing cause of tooth decay is Streptococcus________ 17. A cytoplasmic body that helps a bacterium orient itself. 18.
Nucleic acid forming chromosomes. 19. Many times the length of a bacterium but extremely thin; used for motility. 20. A bacterial sphere. 21. Antibiotic that interrupts
construction of bacterial cell wall. 22. Serves as a buffer between bacterium and external environment. 25. Form displayed by typhoid, anthrax and diphtheria bacilli. 27.
Alternative name of pilus. 29.Bacterium well known for its capsule(initials). 31. Layer of bacteria and other materials on tooth surface. 33. Microscope (abbr) used to
visualize cell surfaces. 34. Used to stain cells for bright field microscopy. 36. Unlike_______ -karyotic cells, bacteria have no nuclei. 38. Common site of infection by
staphylococci. 39. Type of cell(abbr) that cannot easily engulf encapsulated bacteria.
Check your Answers on Page 16
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10
MAY-JUN 2008
Bug of the Month
Salmonella species
The genus Salmonella consists of bacilli that parasitise the
intestines of a large number vertebrate species and infects human
beings, leading to enteric fever, gastroenteritis and septicemia
with or without focal suppuration and the carrier state. The most
important member of the genus is Salmonella typhi, the causative
agent of typhoid fever. In 1880s, the typhoid bacillus was first
observed by Eberth in spleen sections and mesenteric lymph
nodes from a patient who died from typhoid. Robert Koch
confirmed a related finding by Gaffky and succeeded in
cultivating the bacterium in 1881. But due to the lack of
differential characters, separation of the typhoid bacillus from
other enteric bacteria was uncertain. In 1896, it was demonstrated
that the serum from an animal immunized with the typhoid
bacillus agglutinated (clumped) the typhoid bacterial cells, and it
was shown that the serum of patients afflicted with typhoid
likewise agglutinated the typhoid bacillus. Serodiagnosis of
typhoid was thus made possible by 1896.
oral route. Humans normally acquire the disease from eating
contaminated animal products or raw vegetables that have been
contaminated. Other modes of zoonotic transmission include
direct contact with livestock, wildlife, or pets, especially cats and
turtles. Animal-animal transmission happens at the farm through
contaminated food and water sources, pastureland, or contact
with newly acquired animals. On the way to the slaughterhouse,
stress and overcrowded conditions associated with transport
increase the number of bacteria shed. Environmental conditions
at the slaughterhouse can easily cause indirect transmission
through contaminated equipment or people. Since one infected
animal can infect an entire slaughterhouse, and therefore, expose
many people to harmful animal products, safety precautions must
be taken at three different levels, which must cooperate with each
other - the farm, the slaughterhouse, and the preparation of food,
by the consumer/handler.
Antigenic Structure
Salmonellae possess the following antigens based on which they
are classified and identified: (a) Flagellar antigen, H; (b) Somatic
antigen, O; (c) Surface antigen, Vi. H antigen present on the
flagella is a heat labile protein. The H antigen is strongly
immunogenic and induces antibody formation rapidly and in high
titres following infection or immunization. The somatic O
antigen is a phospholipids-polysaccharide complex. It is less
immunogenic than H antigen and the titre of the O antibody
induced after infection or immunization is lower. The Vi antigen
is poorly immunogenic and only low titre antibody is produced
after infection.
Morphology & Cultural Characteristics
Salmonellae are Gram negative rods and motile with peritrichate
flagella. They are aerobic and facultatively anaerobic, growing
readily on simple media over a range of pH 6-8 and temperature
15-41°C. On MacConkey and deoxycholate citrate media,
colonies are colourless due to the absence of lactose fermentation.
On Wilson and Blair bismuth sulphite medium, jet-black colonies
with a metallic sheen are formed due to production of H2S.
Selenite F broth and tetrathionate broth are commonly employed
as enrichment media. Salmonellae ferment glucose, mannitol and
maltose, forming acid and gas. They give Methyl red positive but
Voges Proskauer negative and Citrate negative.
Pathogenicity & Clinical Manifestation
Salmonella infections in humans vary with the serovar, the strain,
the infectious dose, the nature of the contaminated food and the
host status. In the pathogenesis of typhoid the bacteria enter the
human digestive tract, penetrate the intestinal mucosa (causing
no lesion), and are stopped in the mesenteric lymph nodes.
Bacterial multiplication occurs there, and part of the bacterial
population lyses. From the mesenteric lymph nodes, viable
bacteria and endotoxin may be released into the bloodstream
resulting in septicemia. Salmonella excretion by human patients
may continue long after clinical cure. Asymptomatic carriers are
potentially dangerous when unnoticed. About 5% of patients
clinically cured from typhoid remain carriers for months or even
years. Antibiotics are usually ineffective on Salmonella carriage
because the site of carriage may not allow penetration by the
antibiotic.
The term enteric fever includes typhoid fever caused by S. typhi
and paratyphoid fever caused by S. paratyphi. Typhoid is strictly
a human disease. The incidence of human disease decreases when
the level of development of a country increases (i.e., controlled
water sewage systems, pasteurization of milk and dairy products).
Where these hygienic conditions are missing, the probability of
fecal contamination of water and food remains high and so is the
incidence of typhoid. The incubation period is usually 7-14 days
but may range from 3-56 days. The onset is usually gradual, with
headache, malaise, anorexia, a coated tongue and abdominal
Habitats
Salmonellae are disseminated in the natural environment (water,
soil, sometimes plants used as food) through human or animal
excretion. Humans and animals (either wild or domesticated) can
excrete Salmonella either when clinically diseased or after
having had salmonellosis, if they remain carriers. Salmonella
organisms do not seem to multiply significantly in the natural
environment (out of digestive tracts), but they can survive several
weeks in water and several years in soil if conditions of
temperature, humidity, and pH are favorable. The principal
habitat of the salmonellae is the intestinal tract of humans and
animals.
Food can become contaminated with Salmonella during the
slaughter and processing of an animal, when food is handled by a
person infected with Salmonella or from cross-contamination
because of unsanitary food handling practices. The following
listed below have been responsible for foodborne illnesses: raw
and undercooked meat (especially poultry), raw fruits and
vegetables (especially sprouts and cantaloupes) and their juices,
raw and undercooked eggs, unpasteurized dairy products, like
raw milk and raw milk cheeses.
Mode of Transmission
The mode of transmission for S. typhi is water, food borne or
vertical transmission. It is primarily transmitted through fecal
For Private Circulation Only
11
Bug of the Month
bacilli in the patient's sera. The antigens used in the test are H and
O antigens of S. typhi and the H antigens of S. paratyphi A and B.
The detection of carriers is important for epidemiological and
public health purposes. Laboratory tests are also useful in
screening food handlers and cooks to detect carrier state. The
identification of fecal carriers is by isolation of the bacillus from
feces or from bile. For the detection of urinary carriers, repeated
urine cultures should be carried out.
discomfort with either constipation or diarrhea. A soft palpable
spleen is a constant finding. Hepatomegaly is also common. The
most important complications are intestinal perforation,
hemorrhage and circulatory collapse. Relapses may be seen
during the convalescence, particularly when antibiotics treat the
patients. They are due to a re-invasion of the bloodstream from
the tissues in which typhoid bacilli are still proliferating at the
time when the bacteraemic phase of the primary attack is brought
to a close.
Salmonella can also cause food borne intoxication. About 12-24
hours following ingestion of contaminated food (containing a
sufficient number of Salmonella), symptoms appear (diarrhea,
vomiting, fever) and last 2-5 days. Spontaneous cure usually
occurs. Contamination of meat (cattle, pigs, goats, chicken, etc.)
may originate from animal salmonellosis, but most often it results
from contamination of muscles with the intestinal contents
during evisceration of animals, washing, and transportation of
carcasses. When contaminated meat is ground, multiplication of
Salmonella may occur within the ground meat and if cooking is
superficial, ingestion of this highly contaminated food may
produce a Salmonella infection. The incidence of foodborne
Salmonella infection/toxication remains reletavely high in
developed countries because of commercially prepared food or
ingredients for food. Any contamination of commercially
prepared food will result in a large-scale infection.
Egg-associated salmonellosis is an important public health
problem in the United States and several European countries.
Salmonella enteritidis, can be inside perfectly normal-appearing
eggs, and if the eggs are eaten raw or undercooked, the bacterium
can cause illness. A person infected with the Salmonella
enteritidis usually has fever, abdominal cramps and diarrhea
beginning 12 to 72 hours after consuming a contaminated food or
beverage. The illness usually lasts 4 to 7 days, and most persons
recover without antibiotic treatment.
Treatment
Specific antibacterial therapy for enteric fever became available
only in 1948 with the introduction of chloramphenicol. Though S.
typhi is susceptible in vitro to many antibiotics such as
streptomycin and tetracycline, it is ineffective in vivo. Ampicillin,
amoxycillin, furazolidone and cotrimoxazole were the other
drugs that had been found useful in the treatment of typhoid fever.
Some of the strains of Salmonella have become resistant to
chloramphenicol. At present, the drugs useful in treatment of such
multiresistant typhoid cases are the later fluoroquinolones(e.g.
ciprofloxacin, pefloxacin, ofloxacin) and the third generation
cephalosporins (e.g. ceftazidime, ceftrioxone). A combination of
antibacterial therapy along with the vaccine has been tried in the
eradication of carrier state. This combination has also been used
to prevent relapses.
Control Measures
Salmonellae infection can be effectively controlled by general
measures, such as improvements in sanitation and provision of
protected water supply. Many developed countries have been
able to eliminate the risk by these measures. The following
measures should be followed to defeat salmonellae infection:
§ Handwashing is one of the best ways to prevent the spread of
salmonellae related illness. Hands should be washed with
high-level disinfectants after going to the toilet and before
preparing food. Hand washing should be done before
switching from preparing one type of food to another.
§ Kitchen utensils must be properly washed before use with
another type of food. Again, this stops bacteria being
exchanged.
§ Different cutting boards and knives should be used for
preparing different foods.
§ Foods like meat and egg should be cooked thoroughly.
§ Bacteria can grow in the danger zone between 4°C and 60°C.
Food should be kept in refrigerator at or below 4°C.
Refrigeration at or below 4°C slows down most bacterial
growth. Freezing at or below -18°C can stop it completely.
§ Poultry must always be thoroughly cooked or boiled.
§ Minced meat must always be thoroughly cooked or boiled.
§ In most eggs, the salmonella bacteria exist only on the shell.
Eggs should be scalded in boiling water for five seconds
before use.
Diagnosis
Laboratory diagnosis of Salmonella infections depends mainly
on the isolation and identification of the causal Salmonella from a
specimen of the patient's blood, feces, urine or vomit. Testing the
patient's serum for salmonellae antibodies is useful only in the
diagnosis of enteric fever (Widal reaction). For the diagnosis of
pyrexial illnesses blood culture should be done. Salmonella
epidemics may occur among infants in pediatric wards. The
frequency and gravity of these epidemics are affected by hygienic
conditions, malnutrition and the excessive use of antibiotics that
select for multiresistant strains. Bacteremia occurs early in the
disease and blood cultures are positive in approximately 90% of
cases in the first week of fever. When blood samples from a
patient with suspected enteric fever have been submitted for the
Widal test, it is useful, as a routine to cultivate the clot after the
serum has been removed. Salmonellae are shed in the feces
throughout the course of the disease and even in convalescence,
with varying frequency. Fecal cultures are almost as valuable as
blood cultures in diagnosis. Salmonellae are shed in the urine
irregularly and infrequently. A variety of serological tests are
available for the diagnosis of enteric fever like widal
agglutination test, counter immuno electrophoresis, latex
agglutination test, etc. Widal reaction is a test for the
measurement of H and O agglutinins for typhoid and paratyphoid
For Private Circulation Only
MAY-JUN 2008
References
1. Textbook of Microbiology. R.Ananthanarayan & C.K.J.
Paniker. Sixth edition, 2003. Edited by C.K.J. Paniker.
2. Practical Medical Microbiology, Mackie & McCartney, 13th
Edition 1989, Edited by J.G. Collee, J.P. Daguid.
12
MAY-JUN 2008
Did You Know
McFarland Standards
Resistance to a variety of antimicrobial agents is emerging in
bacterial pathogens throughout the world. Since the accuracy of
the antimicrobial susceptibility data is associated with the
performance standard of the test, strict adherence to the standard
procedures is essential. The Kirby-Bauer disc diffusion
susceptibility test, performed in accordance to NCCLS (National
Committee on Clinical Laboratory Standards) method gives
reliable results and hence predicts clinical efficacy of the
antibiotic tested.
The emergence of antimicrobial resistant strains of pathogenic
bacteria has become a great threat to the public health. Early
detection of emerging trends in antimicrobial resistance may
facilitate implementation of effective control measures. The
laboratory testing of antibiotic susceptibility contributes directly
to patient care and the expertise of the microbiology laboratory
can have powerful influence on antibiotic usage and hence on the
pressures that facilitate the emergence of antimicrobial drug
resistance. Standardization of the inoculum in antimicrobial
susceptibility testing by comparison of a broth culture with a
barium sulfate standard has been recommended for routine
performance of both the disc diffusion and the agar dilution test.
2H2O) is added to 99.5 ml of 0.18 mol/L H2SO4 (1% v/v) with
constant stirring to maintain a suspension.
2. The correct density of the turbidity standard should be
verified by using a spectrophotometer with a 1-cm light path
and matched cuvette to determine the absorbance. The
absorbance at 625 nm should be 0.008 to 0.10 for the 0.5
McFarland standard.
3. The Barium Sulfate suspension should be transferred in 4 to 6
ml aliquots into screw-cap tubes of the same size as those used
in growing or diluting the bacterial inoculum.
4. These tubes should be tightly sealed and stored in the dark at
room temperature.
5. The barium sulfate turbidity standard should be vigorously
agitated on a mechanical vortex mixer before each use and
inspected for a uniformly turbid appearance. If large particles
appear, the standard should be replaced. Latex particle
suspensions should be mixed by inverting gently, not on a
vortex mixer
6. The barium sulfate standards should be replaced or their
densities verified monthly.
Now there are McFarland standards prepared from suspensions
of latex particles, which lengthens the shelf life and stability of
the suspensions. The latex standards are prepared by suspending
latex particles in a buffer solution. Sodium azide is added as a
preservative. The standards are adjusted to an acceptable
absorbance range with a one-centimeter light path set at 625nm.
Adjusting bacterial suspension turbidity to a McFarland Standard
produces bacterial counts in an expected range. As with the
barium sulfate standards, a 0.5 McFarland Standard is
comparable to a bacterial suspension of 1.5 X 108 CFU/ml.
McFarland standard 1, 2, 3, 4 & 5are also comparable to 300, 600,
900, 1200 & 1500 millions CFU/ ml respectively.
Principle
Light-scattering techniques have been utilized for many years to
estimate the number of cells in a microbial suspension. The light
scattered by particulate matter during its passage through the
suspension causes the turbidity of a suspension. Accurate
measurements of turbidity and hence bacterial growth can be
obtained in two ways:
§ By measuring the amount of scattered light directly, a
procedure known as nephelometry. This is rarely used in
practice.
§ By measuring the light lost from the beam by scattering, a
procedure known as turbidimetry. This can be measured
easily by using Lambert Beer's law.
Latter method of turbidity estimation is the easiest and quickest
way of calibrating a bacterial population. Standard suspension of
insoluble barium sulphate precipitate described by McFarland
(1907) and Brown (1919-20), which is a series of standards of
different barium sulfate concentrations correspond to
suspensions of different numbers of bacteria/ml.
Original McFarland standards were made by mixing specified
amounts of barium chloride and sulfuric acid together. Mixing the
compounds forms a barium sulfate precipitate, which causes
turbidity in the solution. Other than barium sulfate various ionic
compounds such as calcium carbonate and silver chloride have
been used for many years for turbidity standards. Clays have been
used widely. Representative of these are kaolinite, bentonite,
illite and fuller's earth. These are generally unsatisfactory
because suspensions must be shaken frequently, particle size
changes with time, or turbidity is affected by light.
Application and Interpretation
§ In a tube of the same internal diameter as the standard a
uniform suspension is prepared in saline of bacteria under test
to a density greater than that required.
§ A standard opacity tube of the required density is selected and
shaken it vigorously until all the deposit is raised into uniform
suspension.
§ Bacterial suspension is compared with standard and viewed
with oblique illumination against a dark background.
§ The bacterial suspension is adjusted by dilution with saline
until it matches the standard.
The approximate number of bacteria per milliliter can be
estimated according to the opacity standard. But the numbers
vary with the size of the bacteria.
Precautions
McFarland standard is used for in vitro purpose. It should be
stored at 2-30ºC. It should be stored in original tube and should
not be transferred from original tube.
Turbidity standard for inoculum preparation
To standardize the inoculum density for a susceptibility test, a
BaSO4 turbidity standard, equivalent to a 0.5 McFarland standard
or its optical equivalent (e.g., latex particle suspension), should
be used. A BaSO4 0.5 McFarland standard may be prepared as
follows:
1. A 0.5-ml aliquot of 0.048 mol/L BaCl2 (1.175% w/v BaCl2.
For Private Circulation Only
References
1. Practical Medical Microbiology, Mackie & McCartney, 13th
Edition 1989, Edited by J.G. Collee, J.P. Daguid.
2. William G. Roessler and Carl R. Brewer. 1967. Permanent
Turbidity Standards. Applied Microbiology. 15(5):1114-1121.
13
Best Practices
MAY-JUN 2008
Moist Heat Sterilization By Using Autoclave
by downward displacement, super atmospheric,
transatmospheric or sub-atmospheric pulses.
Factors Influencing Sterilization by Heat
There are some factors, which influence the sterilization:
§ Temperature and time- They are inversely related, shorter
time is sufficient at high temperatures.
§ Sterilization hold time- This is the time for which the entire
load requires to be exposed to pure, dry, saturated steam at the
effective temperature in order to ensure sterilization.
§ Heat penetration time- Before the sterilization hold time can
start, the load needs to be brought up to temperature. This is
the heat penetration time. It varies with the different type of
load.
§ Number of microorganisms and spores-The number of
survivors diminished exponentially with the duration of
heating. It also depends on the species, strains and spore
forming ability of the microbes.
§ Depends on the nature of material- A high content of organic
substances generally tends to protect spores and vegetative
organisms against heat.
Sterilization
is the freeing of an article from all living
organisms, including bacteria and their spores.
Sterilization of culture media, containers and instruments is
essential in microbiological work for isolation and maintenance
of microbes. Sterilization can be done in a variety of ways, which
can be conveniently categorized as follows:
(a) Physical Method- Sterilization can be done by heat, radiation
and filtration. Again heat sterilization can be categorized into
two:
§ Dry Heat
§ Moist Heat
(b) Chemical Methods
Moist Heat Sterilization
Moist-heat sterilization is achieved when water vapour at a
definite temperature is introduced or generated at the level of the
microorganisms to be inactivated and is maintained in such
conditions for a definite time. Moist heat kills the organisms by
coagulating and denaturing their enzymes and structural protein.
Steam contacts a cooler surface, condenses, causing a huge
decrease in volume and setting up a negative pressure that draws
more steam. Condensation occurs as long as there is a
temperature differential. Action of steam ensures: Surface
heating, penetration, and protein coagulation. Sterilization by
moist heat of the most resistant spores generally requires 121 °C
for 15-30 minutes. Moist heat is used for the sterilization of
culture media and all other materials through which steam can
penetrate. Moist heat is more effective than dry heat. Sterilization
can be done at lower temperatures in a given time at a shorter
duration at the same temperature. Moist heat can be employed at
temperatures below 100ºC, temperatures at 100ºC and
temperature above 100ºC. Autoclaving is the most reliable
method to do moist heat sterilization above 100ºC.
Operation Of Autoclave
Autoclave is used for sterilization as well as decontamination.
Basic operating procedures for autoclave are arranging the load,
setting of operating control, sterilization and unloading.
Packaging is an important step for autoclaving. Some of the
following points should be remembered while operating an
autoclave:
§ Volatile chemicals, radioactive compounds and sharps should
not be kept for autoclave.
§ Containers and autoclave bags should be utilized for
autoclaving.
§ Containers should not be overfilled.
§ Packaging should be prepared in such a way that steam can
penetrate easily.
§ Incase of decontamination of biohazardous materials it must
be capped at least with aluminum foils.
§ The material to be autoclaved should not allow touching the
sides or top of the chamber.
Various types of autoclaves are there like: a) Gravity
displacement, b) Vacuum assisted, c) Simple transportable, d)
Porous load. Gravity refers to the way steam enters from the top
of the chamber and forces air out the drain at the bottom. The
objective is to get all the air out of the chamber and replace it with
steam. But in vacuum assisted model use a vacuum to remove air
from the chamber before the steam is allowed to enter. In simple
transportable autoclave the air is removed by turbulent
displacement. In porous load autoclave the air may be extracted
by an oil-seal high vacuum pump or be diluted out by a succession
of steam pulses alternating with the drawing of a partial vacuum.
Packaged and wrapped goods, linen and instruments with lumens
Autoclave
Autoclave is the method most widely used for sterilization of
culture media and surgical supplies. It is a pressurized device
designed to heat aqueous solutions above their boiling point to
achieve sterilization. Charles Chamberland invented it in 1879.
Under ordinary circumstances (at standard pressure), liquid
cannot be heated above 100 °C in an open vessel. However, when
water is heated in a sealed vessel such as an autoclave, it is
possible to heat liquid water to a much higher temperature. As the
container is heated the pressure rises due to the constant volume
of the container. The boiling point of the water is raised because
the amount of energy needed to form steam against the higher
pressure is increased. The standard temperature and pressure for
sterilization in autoclave is 121ºC at 15 lb/inch2. This works well
on solid objects; when autoclaving hollow objects, it is important
to ensure that all of the trapped air inside the hollow
compartments is removed. Autoclaves may achieve air removal
For Private Circulation Only
14
MAY-JUN 2008
Best Practices
where there may be difficulty in removing the air or allowing
sufficient steam penetration, can be effectively processed only in
a porous load or multipurpose autoclave.
characteristics including pressure gauge sensitivity and
response, valve operation, alarm systems functions, timer
response and accuracy, steam flow rates and/or pressures,
cooling water flow rates, cycle controller functions, door
closure gasketing, and air break systems and filters;
§ For new equipment: installation requirements and
installation check points for each system and sub-system;
§ For existing equipment, the necessary upgrading
requirements or any compensatory procedures;
justification for alternate procedures should be available;
§ A description of the following studies like bioburden
determination studies, empty chamber heat distribution
studies, container mapping studies, loaded chamber heat
penetration studies, microbiological challenge studies.
Safety Instructions
Some of the safety instructions should follow while handling an
autoclave:
§ Personal protective equipments (PPE) like laboratory coat,
globes, eye protecting shield; closed toed shoes should be
used during the unloading of autoclave.
§ The door (lid) should be closed tightly during the time of
loading.
§ Chamber pressure should be checked before opening the
autoclave.
§ The door (lid) should be opened slowly to remove the steam.
Indicating devices used in the validation studies or used, as part of
post-validation monitoring or requalification must be calibrated.
Physical and chemical indicators should be tested to demonstrate
adequate predetermined response to both time and temperature.
Detailed written test procedures and records of test results should
be available. The indicators should be used before a written
expiry date and stored to protect their quality. Biological
indicators should be tested according to detailed written
procedures for viability and quantitation of the challenge
organism and for the time/temperature exposure response. For
commercial indicators, a certificate of testing for each lot
indicating the "D" value of the lot should be available. The
biological indicator should be used before expiry and adequately
stored. Following steps should be followed for validation testing:
§ All autoclaves are to be run with cycle log recorders, which
ensure that temperature parameters are achieved. Paper
copies of the results are to be maintained for a period of 1 year.
Any deviations are to be addressed.
§ All loads (sterilization and decontamination) are to include
items labeled with temperature sensitive tape. Any load where
the tape has not changed color will be re-autoclaved with
fresh tape.
§ All autoclaves will have biological indicator testing
performed to confirm efficacy of decontamination cycles.
Bacillus stearothermophilus is used as biological indicator.
Typical biological indicators for combined biological
indicator/bioburden based steam sterilization models include:
B. stearothermophilus, C. sporogenes, B. coagulans and B.
subtilis.
Validation Approaches
The validation of moist heat sterilization processes may be
performed using any of the three strategies outlined below. The
approach selected should be appropriate and adequately
supported.
a) Prospective Validation- This approach applies to new or
modified processes and new equipment. The studies are
conducted, evaluated and the process & equipment system
certified prior to initiating routine production.
b) Concurrent Validation- This approach applies to existing
processes and equipment. Concurrent validation studies are
conducted during regular production and should only be
considered for processes, which have a manufacturing and
testing history indicating consistent quality production.
c) Retrospective Validation- This approach can only be applied
to existing products, processes and equipment and is based
solely on historical information. Normal processing records
generally lack sufficient detail to permit retrospective
validation.
Each stage of the evaluation of the effectiveness and
reproducibility of a sterilization process should be based on a preestablished and approved detailed written protocol. A written
change control procedure should be established to prevent
unauthorized change to the protocol or process and restrict
change during any phase of the studies until all relevant data are
evaluated. The protocol should specify the following in detail:
§ The process objectives in terms of product type, batch size,
container/closure system, and probability of survival
desired from the process;
§ Pre-established specifications for the process, which
include the cycle time, temperature, pressures and loading
pattern;
§ A description of all of the equipment and support systems in
terms of type, model, capacity and operating range;
§ The performance characteristics of each system, subsystem or piece of equipment in performance
For Private Circulation Only
Applications
Autoclaves are found in many medical settings and other places
that need to ensure sterility of an object. Autoclaving is often used
to sterilize medical waste prior to disposal in the standard
municipal solid waste stream.
References
Practical Medical Microbiology, Mackie & McCartney, 13th
Edition 1989, Edited by J.G. Collee, J.P. Daguid.
15
In Focus
In this issue Bug Of the Month article focused on Salmonella
species and Microxpress offers following Kit and dehydrated
culture media for the isolation and identification of Salmonella:
§ Salmonella Identification Kit
A biochemical identification kit contains 12-miniature test
panel.
§ SS Agar
A differential and selective medium for isolation of
Salmonella and some Shigella species from clinical and nonclinical specimens.
§ Tetrathionate Brilliant Green Bile Broth
A medium for isolation and identification of Salmonella.
§ Tetrathionate Bile Brilliant Green Broth IP
A medium for isolation and identification of Salmonella in
compliance with IP.
§ Tetrathionate Bile Brilliant Green Broth (Broth Medium
I) EP
A medium for isolation and identification of Salmonella in
compliance with EP.
§ Tetrathionate Bile Brilliant Green Broth (Broth Medium
I) BP
A medium for isolation and identification of Salmonella in
compliance with BP.
§ Tetrathionate Broth Base Medium USP
A medium for selective enrichment of Salmonella in
compliance with USP.
§ Tetrathionate Broth Base, Hajna
A medium for selective enrichment of Salmonella,
particularly in food and dairy products prior to isolation.
According to the Did You Know article related to McFarland
Standard Microxpress recommends the following kit:
§ McFarland Standard No. 0.5
Turbidity standard, used in antimcrobial susceptibility testing.
and surgical site infections (SSI) were most common nosocomial
infection. An important SSI prevention measure would include
techniques directed at reducing microbial flora by localized skin
prepping at the surgical site.
Our Current Trends topic is related to chlorhexidine gluconate: A
superior antiseptic than povidone iodine. According to our
articles BioShields recommends following antiseptic solutions
for preparation and post closure of skin to prevent the nosocomial
infection.
Skin Preps
l Surgiprep- CHX™
It is an antiseptic solution for preparation and post closure of
skin. Surgiprep-CHX™ can be used in major & minor
surgical procedures. It can also be used in dermatological
applications. It contains 2.5% v/v chlorhexidine gluconate
solution IP, 63% v/v isopropyl alcohol IP and brilliant green as
colour. Suriprep- CHX™ has following features:
§ Rapid Disinfection
§ Degreasing Effect
§ High Residual Effect
§ Quick Drying
§ Effective, Fugitive Skin Marking
§ Few Steps, Good Results
l Surgiprep™
Surgiprep™ contains 2.5% w/v benzalkonium chloride
solution IP, 63% v/v isopropyl alcohol IP and sunset yellow
FCF as colour. It has the same features like Surgiprep-CHX™.
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In Mini Review we have discussed about nosocomial infection,
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Printed and published by D.G. Tripathi, Edited by Amrita Roy for and on behalf of Tulip Diagnostics(P) Ltd., Tulip House, Dr. Antonio Do Rego Bagh, Alto Santacruz, Bambolim Complex,
Post Office Goa, 403 202, India. Fax- 0832 2458544, E-mail: [email protected], Website- www.tulipgroup.com.
Bimonthly Journal Published Exclusively For Tulip Group Customers
For Private Circulation Only
16